The proteasome is a protein complex that is present in all eukaryotic cells, and degrades intracellular proteins that are specifically marked for removal. As such, it regulates cell proliferation, apoptosis, and cell differentiation. In addition, it removes proteins that are misfolded, changed by aging, or damaged by oxidation. Empirical evidence suggests that cancer cells rely more heavily on intact proteasomal function, in part because they divide more frequently and in part because many of their regulatory pathways are disrupted due to aberrant protein structure.
Inhibition of proteasome activity is therefore a therapuetic strategy for treating cancer. For example, the proteasome inhibitor Bortezomib (VELCADE®) has been approved by the FDA and is being used as a first-line therapeutic to treat multiple myeloma and mantle cell lymphoma. Carfilzomib (KYPROLIS®) was approved by the FDA for the treatment of patients with multiple myeloma who demonstrate disease progression after receiving two prior therapies, including Bortezomib and an immunomodulatory agent. Bortezomib as well as new proteasome inhibitors are now in clinical trials to treat different cancers, including hematological and solid tumors.
Current proteasome inhibitors can exhibit significant toxicity, which varies significantly from patient to patient. At present, there is no biomarker assay to monitor the differential impact of proteasome inhibitors on normal vs tumor cell populations, making it difficult to establish a proper therapeutic window, or optimize drug levels in individual patients.
A variety of assays have been used in clinical research to assess either proteasome activity, or measure proteasome activity in blood. The functioning proteasome is a proteolytic complex with chymotrypsin-like, trypsin-like, and peptidyl-glutamyl caspase-like protease activity. Current biochemical assays used to measure proteasome activity utilize quenched fluorogenic peptide substrates that are non-fluorescent until the peptide is cleaved, releasing a highly fluorescent peptide fragment that can be measured using a fluorimeter. The rate of fluorogen appearance can be used to determine the concentration of proteasomal enzymes and their activity. In the presence of a proteasome inhibiting compound (e.g. Bortezomib), generation of fluorescence is inhibited and the rate of fluorescence appearance can be used to measure the effect of inhibitor on proteasome activity.
These assays require a cell lysate because fluorogenic substrates penetrate intact cells poorly, and fluorescent product can easily diffuse out of the cell. It is therefore difficult to differentiate between a small number of target cells containing high concentrations of proteasomes (e.g., cancer cells) and a large number of normal cells containing low concentrations proteasomes. In addition, unless the target cells can first be purified, it is not possible to distinguish the impact of any inhibitor on the target cells as opposed to normal cell populations in a sample. Thus, in order to determine the effectiveness of a proteasome inhibitor in a given patient, the proteasome activity of the total white blood cell extracts from that patient are compared before and after drug administration. As a result, these cell free assays provide little to no information on the functional effectiveness of inhibitors inside a target cell population. In addition, the dilution introduced by cell lysis may cause dissociation of proteasome inhibitors with faster off-rates, making accurate measurement problematic.
There is a need for an assay capable of distinguishing target cells in a cell population and measuring proteasome activity and inhibition thereof by proteasome-specific inhibitors in selected target cells within the cell population. There is also a need for a proteasome activity assay that can be used in whole blood, without requiring cell lysis, thus preventing sample dilution.